Insulin effects on cardiac Na⁺/Ca²⁺ exchanger activity: Role of the cytoplasmic regulatory loop

Insulin can alter myocardial contractility, in part through an effect on the cardiac sarcolemmal Na⁺/Ca²⁺ exchanger (NCX), but little is known about its mechanism of action. The large cytoplasmic domain (f-loop) of NCX is required for regulation by various intracellular factors, and we have shown previously that residues 562-679 are determinants of NCX inhibition by exchanger inhibitory peptide (XIP). Here we show that the same f-loop deletion eliminates the enhancement of NCX current by insulin, and we examine the signal pathways involved in the insulin response. NCX current (I_NCX) was measured in freshly isolated or cultured (up to 48 h) adult guinea pig myocytes and in myocytes expressing canine NCX1.1 with the 562-679 f-loop deletion (NCX-(Δ562-679)) via adenoviral gene transfer. I_NCX was recorded by whole-cell patch clamp as the Ni ²⁺-sensitive current at 37 °C with intracellular Ca²⁺ buffered. Insulin (1μM) increased I_NCX (at +80 mV) by 110 and 83% in fresh and cultured myocytes, respectively, whereas in myocytes expressing NCX-(Δ562-679) the response was eliminated (with 100 μM XIP included to suppress any native guinea pig I_NCX). The insulin effect on I _NCX was not inhibited by wortmannin, a nitricoxide synthase inhibitor, or disruption of caveolae but was blocked by chelerythrine, implicating protein kinase C, but not phosphatidylinositol-3-kinase, in the mechanism. The insulin effect was also not additive with phosphatidylinositol-4, 5-bisphosphate-induced activation of I_NCX. The finding that the 562-670 f-loop domain is implicated in both XIP and receptor-mediated modulation of NCX highlights its important role in acute physiological or pathophysiological regulation of Ca²⁺ balance in the heart.